Malfunctions in the regulation or mechanism of DNA biosynthesis in mammalian cells can result in neoplastic growth. Conversely, detailed knowledge of the enzyme systems active in DNA replication and repair may provide the basis for more effective treatment or prevention of cancer. Therefore, we have chosen to study the regulation and function of enzymes suspected of having key roles in mammalian DNA biosynthesis. Enzymes with DNA-dependent ATP phosphohydrolase activity have functions essential for DNA replication, repair, or recombination in E. coli, and recent data indicate that enzymes with this activity exist in eukaryotic cells. We are proposing in this application to investigate the catalytic properties and biological function of a DNA-dependent ATP phosphohydrolase that we have detected in rat cell extracts. These studies will employ extensively purified enzymes, subcellular DNA synthesis systems, transformed and control rat fibroblasts in culture, and normal rat tissues. We propose to purify sufficient quantities of the DNA-dependent ATP phosphohydrolase to study its physical structure, substrate specificity, and kinetic constants. The purified enzyme will also be tested for the presence of additional enzymatic activities such as those associated with various E. coli DNA-dependent ATP phosphohydrolases, i.e. nuclease, helicase, topoisomerase, stimulation of DNA polymerase, and promotion of D-loop formation. We plan to determine if the intracellular level of the DNA-dependent ATP phosphohydrolase activity changes during the mitotic cell cycle, during induction of DNA repair by carcinogenic chemicals or radiation, or during neoplastic transformation by Rous Sarcoma Virus. Once a functional correlation is established we will use subcellular systems to study the precise role of the DNA-dependent ATP phosphohydrolase in DNA replication or repair.